title: Liposome-based homogeneous luminescence resonance energy transfer.
abstract: There is an increasing need for developing simple assay formats for biomedical screening purposes. Assays on cell membranes have become important in studies of receptor-ligand interactions and signal pathways. Here luminescence energy transfer was studied on liposomes containing europium ion chelated to 4,4,4-trifluoro-1-(2-naphthalenyl)-1,3-butanedione and trioctylphosphine oxide. Energy transfer efficiency was characterized with biotin-streptavidin interaction, and a model assay concept for a homogeneous time-resolved luminescence resonance energy transfer (LRET) assay was developed. Acceptor-labeled streptavidin was bound to biotinylated lipids on the liposomes, leading to close proximity of the LRET pair. The liposome-based LRET assay was optimized for dye incorporation and concentration, biotinylation degree, liposome size, and kinetics. Sensitivity for a competitive biotin assay was at a picomolar range with a coefficient of variation from 7 to 20%. The developed lipid membrane-based system was feasible in separation free LRET assay concept with high sensitivity, indicating that the assay principle can potentially be used for biologically more relevant target molecules.
authors.affiliation: Laboratory of Biophysics, University of Turku, FIN-20520 Turku, Finland. sari.pihlasalo@ut.u.fi
authors.forename: Sari
authors.initials: S
authors.lastname: Pihlasalo
authors.forename: Mika
authors.initials: M
authors.lastname: Hara
authors.forename: Pekka
authors.initials: P
authors.lastname: Hänninen
authors.forename: J Peter
authors.initials: JP
authors.lastname: Slotte
authors.forename: Jouko
authors.initials: J
authors.lastname: Peltonen
authors.forename: Harri
authors.initials: H
authors.lastname: Härmä
mesh_terms: D001360:Avidin / Q000378:metabolism; D001710:Biotin / Q000378:metabolism; D019921:Biotinylation; D004735:Energy Transfer; D004852:Epoxy Compounds / Q000737:chemistry / Q000378:metabolism; D005063:Europium / Q000737:chemistry / Q000378:metabolism; D007659:Ketones / Q000737:chemistry / Q000378:metabolism; D008081:Liposomes / Q000737:chemistry* / Q000378:metabolism; D008163:Luminescent Measurements / Q000379:methods*; D009281:Naphthalenes / Q000737:chemistry / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000737:chemistry / Q000378:metabolism
chemical_list: C534544:4,4,4-trifluoro-1-(2-naphthalenyl)-1,3-butanedione; D004852:Epoxy Compounds; D007659:Ketones; D008081:Liposomes; D009281:Naphthalenes; D009943:Organophosphorus Compounds; D001360:Avidin; D005063:Europium; C007366:diepoxybutane; D001710:Biotin; C044965:trioctyl phosphine oxide